The present invention relates to methods of treating Multiple Sclerosis, particularly Relapsing forms of Multiple Sclerosis.
Multiple Sclerosis
Multiple Sclerosis (MS) is a common neurological disease affecting more than 1 million people worldwide. Its prevalence rate varies between races and geographical latitude, ranging from more than 100 per 100,000 in Northern and Central Europe to 50 per 100,000 in Southern Europe. MS is the most common cause of neurological disability in young and middle-aged adults. Typically, the disease becomes evident before the age of 30 in about 50% of patients; in 25% of the patients the onset of disease is between the ages of 30 to 40, and in 25% the disease appears between the ages of 40 to 50. The female to male ratio is 2:1(4).
MS and the resulting neurological damage have a major physical, psychological, social and financial impact on the patients and on their families. The most common clinical symptoms of MS are paresis, paraesthesia, visual impairment, sexual, bowel, and urinary dysfunction, spasticity, and incoordination. 40 to 50% of patients suffer from cognitive dysfunctions. The extent of neurological deficit, the rate of progression, and the frequency of relapses are highly variable among the affected individuals (2-3).
Most MS patients have a normal life span marked by numerous years of severe progressive disability. The causes of death in patients with MS are respiratory or urinary tract infections rather than the disease itself. There are several distinct types of MS: relapsing-remitting multiple sclerosis (RRMS), which is characterized by unpredictable acute episodes of neurological dysfunction (relapses), followed by variable recovery and periods of clinical stability. 80% to 85% of MS patients are diagnosed with RRMS. More than 50% of the patients having RRMS develop sustained deterioration with or without relapses superimposed; this form is called Secondary Progressive MS (SPMS). Some MS patients developing a progressive deterioration from the beginning can also develop relapses later on; this uncommon form is called primary progressive-relapsing multiple sclerosis (2-3).
Approximately 15% of overall MS patients develop a sustained deterioration of their neurological function from the beginning. This form is known as Primary Progressive MS or PPMS. The diagnosis is currently according to the McDonald's criteria (1). The outcome of a diagnostic evaluation is either “Multiple Sclerosis”, “possible MS” (for those at risk for MS, but for whom diagnostic evaluation is equivocal), or “not MS” (1). Finally, the term clinically isolated syndrome (CIS) applies to those patients who have suffered a first clinical attack but do not comply with the classical diagnostic criteria for definite MS. Nowadays, the presence of new lesions in a second MRI performed at least three months apart is an accepted criterion for a diagnosis of MS in these patients. 10%-20% of patients with an isolated syndrome will not develop MS.
MS is an inflammatory disease that damages the myelin in the Central Nervous System (CNS) causing neurological impairment and, frequently, severe disability. The etiology of MS remains mostly unknown. It is generally assumed that MS is triggered by a combination of autoimmunity, infection and genetic predisposition (2-3). Autoimmune response against myelin components proceeding through the activation of CD4+ T lymphocytes, loss of proper regulation on Th1/Th2 lymphocytes, production of anti-myelin antibodies by B lymphocytes, and possibly, inhibition of CD8+ cytotoxic/suppressor T lymphocytes underlie the pathogenesis of the MS.
MS is characterized by scattered regions of inflammation within the white substance of the CNS, brain and spinal cord. Focal inflammatory events eventually lead to demyelination of the axonal sheaths, degradation of nerve tissue, and finally, to irreversible neurological damage. Although the exact mechanism by which the MS process is initiated remains largely unknown, the target antigens of the autoimmune response in MS are believed to be part of the CNS myelin.
It is unclear whether the different courses of multiple sclerosis described are due to the same or to distinct pathophysiologic processes. Relapses are considered the clinical expression of acute inflammatory focal lesions whereas progression is considered to reflect the occurrence of demyelination, axonal loss and gliosis. Relapsing remitting multiple sclerosis and secondary progressive multiple sclerosis are probably different stages of the same disease while primary progressive multiple sclerosis may imply different processes.
Myelin basic protein (MBP) and proteolipid protein (PLP) are the most common myelin components. Additional, less abundant constituents of myelin, such as myelin associated glycoproteins (MAG), myelin oligodendrocyte glycoproteins (MOG), and α-B crystalline are also known. There is a considerable on-going debate regarding the true nature of the target antigen(s) in multiple sclerosis. In general, it appears that involvement of different antigens leads to certain differences in the courses of the disease (5-12).
MRI-Based Multiple Sclerosis Diagnostic Criteria
All the diagnostic criteria for establishing the diagnosis of MS proposed in the last 50 years are based on three main principles: (1) demonstration of demyelinating lesions disseminated in space (DIS); (2) demonstration of demyelinating lesions disseminated in time (DIT); and (3) reasonable exclusion of alternative explanations for the clinical presentation (13).
Conventional MRI
Conventional MRI techniques (cMRI), such as T2-weighted sequences and gadolinium-enhanced T1-weighted sequences, are highly sensitive for detecting MS plaques and can provide quantitative assessment of inflammatory activity and lesion load.
MRI studies in patients with Relapsing Remitting Multiple Sclerosis (RRMS) and Secondary Progressive Multiple Sclerosis (SPMS), using gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA) as a contrast agent, indicating blood-brain barrier disruption, have revealed that disease activity (defined as the presence of Gd-enhancing lesions on T1-weighted MRI) is 5 to 10 times more frequent than is apparent from clinical criteria alone (14).
Non-Conventional MRI
Unenhanced T1-weighted imaging, measures of central nervous system atrophy, magnetization transfer imaging, proton magnetic resonance spectroscopy, diffusion-weighted imaging, and functional magnetic resonance imaging, provide a better approximation of the pathological substrate of the multiple sclerosis plaques, have increased the understanding of the pathogenesis of the disease, and have proven useful for studying the natural history of multiple sclerosis and monitoring the effects of new treatments (13).
Current Therapeutic Approaches
The vast majority of treatments for multiple sclerosis have either immunosuppressive or immunomodulatory nature.
Corticosteroids:
Corticosteroids shorten the duration of a relapse but do not generally affect the degree of recovery. They have unspecific immunomodulatory and anti-inflammatory effects that decrease the blood-brain barrier (BBB) permeability, reduce edema, and improve axonal conduction. Corticosteroids are often used to treat clinically significant relapses for a faster recovery. They have an acute anti-inflammatory activity which is short term and does not have an effect on the long term disease course.
Interferon Beta:
Two forms of recombinant interferon beta, beta-1a and beta-1b, have been approved for the treatment of patients with RRMS. The mechanism of their action in MS includes anti-proliferative effects on T lymphocytes; decreased expression of major histocompatibility complex (MHC) class II antigens, and other immunoregulatory properties. The interferon-beta reduces the relapse rate in about 30% of patients with RRMS with mild to moderate disability. Major reduction in Gd-enhancing lesions on MRI scans could be demonstrated with interferon beta-1b, as well as reduction of EDSS progression. Interferon beta is administered as subcutaneous (s.c.) or intramuscular (i.m.) injection once or three times per week, depending on its type. It is associated with various adverse events, including flu-like symptoms, injection site reactions, serum aminotransferase elevation, and depression. Neutralizing antibodies have been reported in 5% to 40% of patients treated for three years and may lead to decreased efficacy.
Glatiramer Acetate:
Glatiramer acetate is a complex of synthetic peptides resembling myelin basic protein, which has shown a reduced annual rate of relapses of 30% in patients with RRMS. Glatiramer acetate is given daily by s.c. injection. The most common side effect is injection site reaction, which has been reported in up to 90% of patients. Another uncommon adverse event is a complex of immediate post injection reactions (IPIRs) which includes flushing, chest tightness, shortness of breath, palpitations, and anxiety.
Natalizumab:
Natalizumab is a humanized monoclonal antibody against the cellular adhesion molecule α4-integrin. Natalizumab is used in the treatment of multiple sclerosis and Crohn's disease. It is co-marketed by Biogen Idec and Élan as Tysabri, and was previously named Antegren. Natalizumab is administered by intravenous infusion every 28 days. The drug is believed to work by reducing the ability of inflammatory immune cells to attach to and pass through the cell layers lining the intestines and blood-brain barrier. Natalizumab has proven effective in treating the symptoms of both diseases, preventing relapse, vision loss, cognitive decline and significantly improving quality of life in people with multiple sclerosis, as well as increasing rates of remission and preventing relapse in Crohn's disease. Natalizumab was approved in 2004 by the United States Food and Drug Administration. It was subsequently withdrawn from the market by its manufacturer after it was linked with three cases of the rare neurological condition progressive multifocal leukoencephalopathy (PML) when administered in combination with interferon beta-1a, another immunosuppressive drug often used in the treatment of multiple sclerosis. After a review of safety information and no further deaths, the drug was returned to the US market in 2006 under a special prescription program. In the European Union, it has been approved only for the treatment of multiple sclerosis.
Antisense Theory
Antisense Oligonucleotides (AS-ONs) are short stretches of nucleotides or nucleotide derivatives that are complementary to a region of targeted RNA and can specifically suppress expression and other aspects such as processing of that particular transcript. The exact mechanism(s) of AS-ON action remains unclear, but is known to be different depending on the type of AS-ONs. Generally, these molecules block gene expression by hybridizing to the target mRNA, resulting in subsequent double-helix formation. This process can occur at any point such as transcription, initiation of translation, or during translation. Some of the possible mechanisms are disruption of splicing, impaired mRNA transport, disruption of translation of the transcripts as well as decreased stability of the mRNA transcript. In the case of many antisense oligodeoxyribonucleotides (AS-ODNs), cellular RNase H is able to bind to the DNA-RNA duplex and hydrolyze the RNA, resulting in reduced transcript numbers and decreased production of protein. Modifications to the deoxy moiety at the 2′-sugar position usually prohibits RNase H recruitment and action in that region of an AS-ODN (16).
Modified AS-ONs or AS-ON analogs are often employed for in vivo antisense applications due to their increased stability and nuclease resistance. A longer serum half-life ensures that the AS-ON has ample time to reach and interact with its target RNA in the tissue. AS-ODNs with phosphorothioate backbones are widely used due to their longer serum half-life and the fact that they are a suitable RNase H substrate. However, phosphorothioates display high affinity for various cellular proteins, which can result in sequence-nonspecific effects. Many AS-ONs with 2′-modifications of the sugar with groups such as O-methyl, fluoro, O-propyl, O-allyl, or many others exhibit greater duplex stability with their target mRNA and greater specificity but antisense effects in that 2′ modified region are usually independent of RNase H. These modifications create bulk at the 2′ position, causing steric hindrance to play a significant role in increasing nuclease resistance. Nucleotide analogs, such as peptide nucleic acids, generally are also nuclease-resistant and often demonstrate superior hybridization properties due to modified backbone charge, although they usually are not acceptable substrates for RNase H (16).
The traditional goal of the antisense approach to therapeutics is to decrease the level of key proteins in the disease pathogenesis. The use of antisense oligonucleotides as therapeutics has the potential advantage of much greater specificity compared to conventional small molecule drugs. The majority of drugs currently in use modulate the activity of specific proteins by either binding directly to the protein of interest or by binding to other proteins, such as cell surface receptors, which then modulate the target protein. Due to the large number of related proteins, activity classes and protein families performing the same or very similar function, small molecule drugs often bind to, and affect the activity of, more than one target protein. In contrast, the effectiveness of AS-ONs relies on highly specific base-pairing between the oligonucleotide and the target RNA. Therefore, antisense technology enables targeting of a single member of a closely-related protein family and designing therapeutic agents displaying fewer non-specific toxic effects than other, less selective, agents (17-21).
VLA-4 Integrin
Integrins are heterodimeric adhesion molecules that play key roles in leukocyte activation, trafficking, and signaling. The VLA-4 integrin consists of α4 chain non-covalently linked to the β1 subunit. It is expressed on most leukocytes, whether they occur in peripheral blood, lymphoid tissue, or at sites of inflammation in various organs. α4β1 binds to VCAM-1 on the activated endothelium and to the CS1 segment of fibronectin found in extracellular matrix. These interactions are critical for leukocyte migration across endothelium and into inflamed tissues. Ligand binding by α4 integrins has diverse biological consequences. The best-known role for α4 is its function as an adhesion molecule guiding leukocytes across vascular endothelium and into sites of inflammation. Leukocytes are recruited from the blood and into tissues by a multi-step process that involves an initial transient rolling of cells along the vascular endothelium followed by firm adhesion and subsequent trans-endothelial migration. The α4 integrin is unique among adhesion molecules in that it can support both the rolling and firm adhesion steps (22).